EP4139484A1 - Procédés thérapeutiques pour la prévention des métastases et des récidives tumorales - Google Patents

Procédés thérapeutiques pour la prévention des métastases et des récidives tumorales

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Publication number
EP4139484A1
EP4139484A1 EP21791579.2A EP21791579A EP4139484A1 EP 4139484 A1 EP4139484 A1 EP 4139484A1 EP 21791579 A EP21791579 A EP 21791579A EP 4139484 A1 EP4139484 A1 EP 4139484A1
Authority
EP
European Patent Office
Prior art keywords
mitochondrial
gene signature
tumor
patient
metastasis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21791579.2A
Other languages
German (de)
English (en)
Other versions
EP4139484A4 (fr
Inventor
Michael P. Lisanti
Federica Sotgia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lunella Biotech Inc
Original Assignee
Lunella Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lunella Biotech Inc filed Critical Lunella Biotech Inc
Publication of EP4139484A1 publication Critical patent/EP4139484A1/fr
Publication of EP4139484A4 publication Critical patent/EP4139484A4/fr
Pending legal-status Critical Current

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    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
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    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Definitions

  • the present disclosure relates to pharmaceutical compounds and companion diagnostics for treating and preventing cancer metastasis, recurrence, and Tamoxifen resistance, in breast cancer.
  • CSCs cancer stem cells
  • cancer cell mitochondria As a new promising therapeutic target for the eradication of CSCs.
  • New evidence suggests that CSCs have elevated levels of mitochondrial biogenesis that helps to energetically drive their rapid propagation and anchorage-independent growth.
  • metastatic breast cancer cells in positive lymph nodes, removed from patients, show a significant increase in mitochondrial Complex IV activity, as seen by histochemical- and immuno-staining.
  • Mitochondrial biogenesis is strictly dependent on the function of the mitochondrial ribosome, which consists of both large and small subunits, to effectively carry out the mitochondrial protein translation of 13 key genes that are absolutely required for OXPHOS and mitochondrial ATP production.
  • mitochondria originally evolved from engulfed aerobic bacteria, an event estimated to have occurred approximately 1.5 billion years ago.
  • certain FDA-approved drugs inhibit mitochondrial protein translation as an off-target side effect.
  • Doxycycline a Tetracycline family member
  • Azithromycin an Erythromycin family member
  • Inhibiting mitochondrial protein translation has been demonstrated as an effective approach for inhibiting CSC propagation in a wide variety of cancer types. Both Doxycycline and Azithromycin effectively inhibit the anchorage-independent propagation of CSCs, as assessed using the 3D-tumor-sphere assay, in cell lines derived from 8 different cancer types, including breast cancers (MCF7, T47D, MDA-MB-231 and MCF10.DCIS.COM).
  • Mitochondrial biogenesis inhibitors are effective against a wide variety of cancer types.
  • CSCs Cancer stem cells, or CSCs
  • MCF7 2D-monolayers with 3D-mammospheres, which are enriched in CSCs.
  • the inventors observed that 25 mitochondrial-related proteins were > 100-fold over-expressed in 3D- mammospheres in a large collection of transcriptional profding data from ER(+) breast cancer patients.
  • a 4-gene signature may be used for predicting distant metastasis in breast cancer patients.
  • measuring the levels of expression of the genes may involve measuring the level of expression of mRNA.
  • measuring the levels of expression of the genes involves measuring the levels of expression of the proteins encoded by the genes.
  • gene expression levels may be measured as increased expression levels of the genes relative to a control.
  • the gene signatures described herein may be used to identify candidates for mitochondrial inhibition therapy.
  • mitochondrial inhibitors As described herein, a series of mitochondrial inhibitors, previously shown to target mitochondria and selectively inhibit 3D-mammosphere formation in MCF7 cells and cell migration in MDA-MB-231 cells, were demonstrated to prevent and inhibit metastasis and recurrence.
  • the five demonstrative mitochondrial inhibitors evaluated showed preferential and selective inhibition of tumor cell metastasis, without causing significant toxicity.
  • all five of the demonstrative mitochondrial inhibitors have been previously shown to induce ATP-depletion in cancer cells. It should be appreciated that other mitochondrial inhibitors may be used for mitochondrial inhibition therapy, without departing from the present approach.
  • MRPL large mitochondrial ribosomal proteins
  • candidates may receive a pharmaceutically effective amount of a mitochondrial inhibitor, such as the Mitoriboscin compounds, Bis-TPP, and Dodecyl-TPP discussed herein.
  • a mitochondrial inhibitor such as the Mitoriboscin compounds, Bis-TPP, and Dodecyl-TPP discussed herein.
  • Embodiments of the present approach may take the form of methods for preventing and/or reducing the likelihood of tumor metastasis and tumor recurrence in a patient.
  • a biological sample of a cancer from the patient may be obtained, and the expression level of genes in one or more gene signatures may be determined.
  • the level of at least one mitochondrial biomarker in the biological sample of a CSC-based mitochondrial- related gene signature comprising ACLY, VDAC3, HADH2, COX6B1, ATP5B, MCCC1, SLC25A10, TIMM8A, ECH1, ACACA, HSPA9, CHCHD2, and CCDC47.
  • the determined levels may be compared to a control or threshold level for the biomarkers. If the determined level exceeds the threshold level, then the patient providing the biological sample may be identified as a candidate for mitochondrial inhibitor therapy, and may be administered a pharmaceutically effective amount of at least one mitochondrial biogenesis inhibitor.
  • the level of each gene in a gene signature may be evaluated. For example, each biomarker in the CSC-based mitochondrial-related gene signature of ACLY, VDAC3, HADH2, COX6B1, ATP5B, MCCC1, SLC25A10, TIMM8A, ECH1, ACACA, HSPA9, CHCHD2, and CCDC47, may be determined.
  • the CSC-based mitochondrial-related gene signature comprises each of ACLY, VDAC3, HADH2, and COX6B 1.
  • the gene signature may be comprised of large mitochondrial ribosomal proteins (MRPL).
  • the gene signature may comprise one or more, or each, of MRPL 15, MRPL13, MRPL 17, MRPL46, MRPL 18, MRPL48, MRPL3, MRPL24, and MRPL4.
  • the patient and potential candidate may be a patient receiving hormone therapy, and the gene signature may include each of MRPL 15, MRPL46, MRPL17, MRPL24, MRPL18, and MRPL13.
  • the patient and potential candidate may be a patient receiving hormone therapy, and the gene signature may include each of MRPL15, MRPL3, MRPL 17, MRPL 18, MRPL24, MRPL13, MRPL48, and MRPL46.
  • the large mitochondrial ribosome-related gene signature may include one or more, or each of, MRPL42, MRPL41, MRPL54, MRPL13, MRPL36, and MRPL22.
  • the present approach may take the form of a kit having the reagents for detecting expression levels of the genes in one or more of the gene signatures described herein.
  • the kit may have nucleic acid probes that specifically bind to nucleotide sequences corresponding to the genes in one or more of the gene signatures described, and a means of labelling the nucleic acids.
  • the kit may have antibodies or ligands that specifically bind to polypeptides encoded by the genes in one or more of the gene signatures described herein, and a means of labelling the antibodies or ligands that specifically bind to polypeptides or peptides encoded by the genes
  • a patient whose gene expression levels are elevated relative to a control or a threshold value may be classified as a candidate for mitochondrial inhibitor therapy, and administered a pharmaceutically effective amount of a mitochondrial inhibitor.
  • mitochondrial inhibitors include tetracycline, doxycycline, tigecycline, minocycline, eyrthromycin, azithromycin, clarithromycin, pyrvinium pamoate, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, berberine, stiripentol, chloroquine, etomoxir, perhexiline, a mitoriboscin, a mitoketoscin, a mitoflavoscin, a mitoflavin, a TPP- derivative, an mDIVIl-1 derivative, caffeic acid phenyl ester, an antimitoscin, and a repurposc
  • Figure 1 shows a Kaplan-Meier curve forthe CSC-based mitochondrial 13-gene signature.
  • Figures 2A and 2B show Kaplan-Meier curves for the CSC-based mitochondrial
  • Figure 3 shows the effects of the three Mitoriboscins (23/G4, 24/D4, 24/F9) and
  • Figure 4 shows the effects of the three Mitoriboscins (23/G4, 24/D4, 24/F9) and
  • Figure 5 shows the effects of Mitoriboscin compound 23/G4 on tumor growth.
  • Figure 6 shows the effects of Mitoriboscin compound 23/G4 on cancer metastasis.
  • Figure 7 shows the effects of Dodecyl-TPP on tumor growth.
  • Figure 8 shows the effects of Dodecyl-TPP on cancer metastasis, at the same micro-molar concentrations.
  • Figures 9A and 9B show Kaplan-Meier curves for the 9-gene, large mitochondrial ribosome signature, as a predictor of distant metastasis and tumor recurrence, respectively, in ER(+) breast cancer patients.
  • Figures 10A and 10B show Kaplan-Meier curves demonstrating that this large mito-ribosome gene signature predicts distant metastasis and tumor recurrence, respectively, in ER(+) breast cancer patients, treated with Tamoxifen.
  • Figures 11A and 11B show the Kaplan-Meier curves for the 6-gene, large mitochondrial ribosome gene signature for predicting distant metastasis and tumor recurrence, respectively, in ER(-) / basal breast cancer patients.
  • Figure 12 shows the Kaplan-Meier curves for the 6-gene signature for predicting overall survival in ER(-)/basal breast cancer patients.
  • salt of a compound relates to corresponding salt prepared by using acid selected from the group of mineral acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulphuric acid, and organic acids, such as tartaric acid, acetic acid, trifluoroacetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid and succinic acid, and alkylsulphonic acids such as methanesulphonic, ethanesulphonic acids, ethane- 1,2-disulfonic acid and 2- hydroxyethanesulfonic acid and arylsulphonic acids such as benzene sulfonic acid, 2- naphthalenesulfonic acid, p-toluenesulphonic acid and naphthalene- 1,5-disulfonic acid.
  • mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric
  • phrases, “pharmaceutically effective amount” as used herein indicates an amount necessary to administer to a host, or to a cell, tissue, or organ of a host, to achieve a therapeutic result, such as the regulating, modulating, or inhibiting protein kinase activity, e.g., inhibition of the activity of a protein kinase, or treatment of cancer.
  • a therapeutic result such as the regulating, modulating, or inhibiting protein kinase activity, e.g., inhibition of the activity of a protein kinase, or treatment of cancer.
  • the person having an ordinary level of skill in the art can use known methods to determine the pharmaceutically effective amount for a given compound and a candidate in need of treatment.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • common and known methods in the art may be used to establish both the maximum tolerable dose of a compound, and the effective dose that provides a detectable therapeutic benefit to a person in need thereof.
  • common and known methods in the art may be used to determine the dosage and dosing schedule for administering the therapeutic agent sufficient to provide a detectable therapeutic benefit.
  • the demonstrative dosing examples disclosed herein in no way limit the potential dosage and dosing schedules that may be provided under the present approach.
  • the term “about” means having a value falling within an accepted standard of error of the mean, when considered by one of ordinary skill in the art. As would be expected, the meaning of “about” depends on the context in which it is used. Frequently, the term “about” may refer to ⁇ 5%, and preferably ⁇ 2.5%, and more preferably ⁇ 1% of the value or range to which it refers. For example, in the context of weight fractions, the phrase “about 20%” may mean 20% ⁇ 5%, preferably 20% ⁇ 2.5%, and more preferably 20% ⁇ 1%. In the absence of specific reference, the term “about” denotes ⁇ 5% of the stated value.
  • the terms “treat,” “treated,” “treating,” and “treatment” include the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated, in particular, cancer.
  • the treatment comprises diminishing and/or alleviating at least one symptom associated with or caused by the cancer being treated, by the compound of the invention.
  • treatment can be diminishment of one or several symptoms of a cancer or complete eradication of a cancer.
  • control typically refers to a sample, reference, or standard that is used as a basis for comparison with one or more experimental or test samples.
  • An experimental sample can be a tumor specimen or sample obtained from a patient.
  • the control may be, for example, a sample that is obtained from a healthy individual free of cancer or tumors.
  • the control may be a non-tumor tissue sample taken from the individual having the cancer or tumor, such as healthy breast tissue.
  • the control may also be a standard reference value, or a range of values, or a historical control.
  • a standard range of values may be obtained from a previously tested control sample, e.g., a group of samples that represent baseline or normal values, such as the levels of the genes of non-tumor breast tissue; or a previously-tested group of individuals who experienced cancer recurrence or metastasis, or did not experience cancer recurrence or metastasis.
  • controls that can serve as standards of comparison to a test sample for the determination of differential gene expression include samples that are believed to be normal, such as from a subject who does not have a cancer or tumor.
  • a range of values, such as laboratory values or values obtained from in vitro experiments, may also be used as a control.
  • a control can be a relative amount of gene expression in a biological sample, or test population.
  • CSCs are the etiological cause of treatment failure in most cancer patients, as they are the cellular drivers of tumor recurrence, metastasis and drug -resistance.
  • new therapeutic approaches are needed to effectively eliminate CSCs.
  • the inventors previous studies identified CSC mitochondria as a potential new therapeutic target. More specifically, the inventors experimentally observed that MCF7-derived 3D-mammospheres are specifically enriched in mitochondrial proteins; 25 mitochondrial proteins showed greater than 100-fold over-expression, while 9 of these proteins were infinitely up-regulated, as compared with 2D-monolayers.
  • N l,395 patients
  • This clinical evidence supports the understanding that CSC mitochondria may play a critical functional role in the metastatic dissemination of cancer cells.
  • CAM chorio-allantoic membrane
  • mitochondrial inhibitors including the Mitoriboscins, have been previously described to effectively inhibit 3D-mammosphere formation in MCF7 cells and cell migration in MDA-MB-231 cells. All five of these mitochondrial inhibitors selectively prevented MDA-MB-231 tumor metastasis, but had only minor effects or no effect on tumor formation. These studies also provide the necessary in vivo functional evidence, that mitochondrial inhibitors can successfully prevent cancer metastasis. These findings could have important clinical implications, for ultimately preventing treatment failure in breast cancer patients.
  • Mitoriboscin compounds showed minor effects on initial tumor growth, but a functional prevention of cancer metastasis.
  • data described herein shows that Mitoriboscin compound 23/G4 had a minor effects on tumor growth, while Mitoriboscin compounds 24/D4 and 24/F9 had no inhibitory effects on tumor growth.
  • all three of these compounds functionally prevented cancer metastasis.
  • Quantitatively similar results were obtained with another independent class of mitochondrial inhibitors, referred to as TPP-Derivatives and described in International Application Publication WO 2019/104115 Al, filed November 21, 2018 and incorporated by reference in its entirety.
  • TPP -Derivative compounds butene- 1,4-bis-triphenyl-phosphonium (Bis-TPP) and dodecyl-triphenyl-phosphonium (Dodecyl-TPP) showed functional prevention of cancer metastasis.
  • Bis-TPP and Dodecyl-TPP both contain a TPP moiety, which functions as a chemical signal for mitochondrial targeting.
  • cancer stem cells After a breast cancer diagnosis, most patients undergo surgical resection of the primary tumor and are then subsequently treated with hormone-, chemo- and/or radio-therapy, depending on the breast cancer subtype. However, many patients ultimately experience treatment failure, resulting in tumor recurrence and distant metastasis. Unfortunately, distant metastasis is responsible for the premature deaths in the vast majority of cancer patients, approaching over 90%. Therefore, new diagnostics and therapeutics are urgently needed to prevent and treat metastatic disease, which has been attributed to the existence and resurgence of a small sub-population of cancer cells, known as cancer stem cells or CSCs.
  • CSCs cancer stem cells
  • the inventors previously carried out unbiased proteomics analysis on MCF7 cell 2D- monolayers, directly compared with MCF73D-mammospheres, as mammospheres are known to be highly enriched in CSCs and progenitor cells. As a consequence, the inventors observed that 25 mitochondrial proteins were highly up-regulated by over 100-fold, specifically in 3D- mammospheres.
  • the inventors interrogated whether the mRNA transcripts of these mitochondrial proteins show any prognostic value in large numbers of ER(+) human breast cancer patients.
  • 13 gene transcripts showed prognostic value in predicting distant metastasis.
  • These genes are ACLY, VDAC3, HADH2, COX6B1, ATP5B, MCCC1, SLC25A10, TIMM8A, ECH1, ACACA, HSPA9, CHCHD2, and CCDC47.
  • Figure 1 shows a Kaplan-Meier curve for the CSC-based mitochondrial 13-gene signature.
  • the 13 -gene signature predicts distant metastasis in ER(+) breast cancer patients.
  • Table 3, below, shows the prognostic value of mitochondrial-related proteins up-regulated in MCF7 Mammospheres, Evaluated in ER(+) Breast Cancer Patients (DMFS/ER(+)/N l,395/>240- months).
  • Figures 2A and 2B show Kaplan-Meier curves for the CSC-based mitochondrial
  • the 4-gene signature predicts distant metastasis and tumor recurrence in ER(+) breast cancer patients.
  • the gene signatures described herein may be used to identify candidates for treatment with mitochondrial inhibitors, i.e., mitochondrial inhibition therapy. As one having an ordinary level of skill in the art will appreciate, a candidate showing over-expression of the genes in the gene signatures above are more likely to benefit from mitochondrial inhibition therapy. The following paragraphs describe examples of mitochondrial inhibitors that may be used in candidates for mitochondrial inhibition therapy.
  • Mitoriboscins a series of mitochondrial inhibitors that were previously developed to specifically target the propagation of CSCs, known as Mitoriboscins. These inhibitors were developed via in silico screening of a library of 45,000 compounds, to identify positive hits that bound to the 3D-structure of the large mitochondrial ribosome. After 880 positive hits were identified, these compounds were then subjected to phenotypic drug screening, using an ATP -depletion assay, and directly validated using the Seahorse Metabolic Flux analyser, to confirm their specificity as bona fide mitochondrial inhibitors.
  • the inventors also evaluated the activity of another mitochondrial inhibitor, namely butene- 1,4-bis-triphenyl-phosphonium (Bis-TPP), which was identified as an inhibitor of 3D- mammosphere formation in MCF7 cells, with an IC-50 of less than 0.5 mM.
  • Bis-TPP butene- 1,4-bis-triphenyl-phosphonium
  • FIG. 3 shows the effects of the three Mitoriboscins (23/G4, 24/D4, 24/F9) and Bis-TPP on MDA-MB-231 tumor growth in the CAM assay.
  • the four inhibitors showed minor effects on tumor growth in the CAM assay, as a result of the 8-day period of drug administration. Note that these results do not indicate that these compounds are ineffective at inhibiting CSCs - the bulk cancer cells are already forming in the CAM assay at the time of drug administering, and further evaluations are planned using increased concentration of the compounds.
  • MDA-MB-231 cells and the CAM assay in chicken eggs were used to quantitatively measure spontaneous tumor mestastasis.
  • An inoculum of 1 x 10 6 MDA-MB- 231 cells was added onto the CAM of each egg (on day E9) and then eggs were then randomized into groups.
  • tumors were detectable and they were then treated daily for 8 days with vehicle alone (1% DMSO in PBS) or the four mitochondrial inhibitors.
  • the lower CAM was collected to evaluate the number of metastatic cells, as analyzed by qPCR with specific primers for Human Alu sequences. The results are summarized in Figure 4.
  • FIG 4 illustrates that all three Mitoriboscins were clearly effective in inhibiting metastasis.
  • Mitoriboscin compounds 24/D4 and 24/F9 were the most effective of the Mitoriboscins, and Bis-TPP also significantly prevented metastasis.
  • All four mitochondrial inhibitors tested showed significant effects on MDA-MB-231 metastasis. The same procedure may be used to evaluate the anti-metastasis and anti-recurrence effects of other mitochondrial inhibitors.
  • Mitoriboscin compound 23/G4 was minimally effective at a concentration of 0.5 mM, it was tested at higher concentrations of 0.75 mM, 1 mM, and 2 mM.
  • Figure 5 shows the effects of Mitoriboscin compound 23/G4 on tumor growth. As can be seen, compound 23/G4 inhibited tumor growth by 40% to 60% at the higher concentrations (averages are shown + SEM. ***p ⁇ 0.001).
  • Figure 6 shows the effects of Mitoriboscin compound 23/G4 on cancer metastasis. As expected, the effects of compound 23/G4 on metastasis were significantly more pronounced. At the higher concentrations tested, compound 23/G4 significantly inhibited metastasis by about 70-75%. The inhibition effects of compound 23/G4 on metastasis were significantly more pronounced than its effects on tumor growth. Averages are shown + SEM. ***p ⁇ 0.001.
  • FIG. 7 shows the effects of Dodecyl-TPP on tumor growth.
  • Dodecyl- TPP significantly inhibited tumor growth by 12% to 40% (averages are shown + SEM. *p ⁇ 0.05; ***p ⁇ 0.001).
  • the structure of Dodecyl-TPP (d-TPP) is below. Note the 12-carbon alkyl -chain attached to the lipophilic cation, triphenyl-phosphonium (TPP).
  • Figure 8 shows the effects of Dodecyl-TPP on cancer metastasis, at the same micro-molar concentrations.
  • Dodecyl-TPP significantly inhibited metastasis by 25% to 65% (averages are shown + SEM. *p ⁇ 0.05; ***p ⁇ 0.001). Little or no toxicity was observed for Dodecyl-TPP at 6.25 mM and 25 mM, as evident from Table 6, above.
  • Dodecyl-TPP preferentially targeted metastasis, rather than tumor growth.
  • Dodecyl- TPP showed some toxicity 62.5 mM, preventing reliable analysis of its effects on tumor growth and metastasis, at this higher concentration.
  • the inventors Given the functional effects of the Mitoriboscin compounds on metastasis, the inventors also evaluated if the gene mRNA transcripts of the large mitochondrial ribosomal proteins (MRPL) show any prognostic value in ER(+) and ER(-)/basal breast cancer patients.
  • MRPL mitochondrial ribosomal proteins
  • This gene signature included MRPL15, MRPL13, MRPL17, MRPL46, MRPL18, MRPL48, MRPL3, MRPL24, and MRPL4. Table 7, below, shows the results of this
  • FIGS 9A and 9B show Kaplan-Meier curves for the 9-gene, large mitochondrial ribosome signature, as a predictor of distant metastasis and tumor recurrence, respectively, in ER(+) breast cancer patients.
  • This gene signature includes MRPL15, MRPL46, MRPL17, MRPL24, MRPL18, and MRPL13.
  • FIGs 10A and 10B show Kaplan-Meier curves demonstrating that this large mito-ribosome gene signature predicts distant metastasis and tumor recurrence, respectively, in ER(+) breast cancer patients, treated with Tamoxifen.
  • This signature includes MRPL42, MRPL41, MRPL54,
  • FIGs 11A and 11B show the Kaplan-Meier curves for the 6-gene, large mitochondrial ribosome gene signature for predicting distant metastasis and tumor recurrence, respectively, in ER(-) / basal breast cancer patients.
  • the same 6-gene signature may also be used as a predictor of overall survival in ER(-) / basal breast cancer patients.
  • Figure 12 shows the Kaplan-Meier curves for the 6-gene signature for predicting overall survival in ER(-)/basal breast cancer patients.
  • the mitochondrial biogenesis inhibitor is administered if the determined level for all three biomarkers exceeds the threshold level.
  • the threshold level for each biomarker in the Mito-Signature may be determined using a non-cancerous epithelial sample from the same subjectlf a patient exhibits elevated expression of the genes in a signature, then the patient may receive a pharmaceutically-effective amount of a mitochondrial inhibitor, such as the Mitoriboscin compounds described herein, Bis-TPP, or Dodecyl-TPP, as non-limiting examples.
  • Mitochondrial inhibition is an effective strategy for inhibiting cancer recurrence and metastasis, and for eradicating cancer cells and CSCs in particular.
  • a number of categories of mitochondrial inhibitors may be used in connection with the present approach.
  • a first category of mitochondrial inhibitors are Mitoriboscins, as described above and in U.S. Patent 10,512,618, issued December 24, 2019 and incorporated by reference in its entirety.
  • a second category of mitochondrial inhibitors include combination therapies involving oxidative metabolism inhibitors and glycolytic metabolism inhibitors.
  • International Application No. PCT/US2018/028587 filed April 20, 2018 and published as WO 2018/195434-A1, is incorporated by reference in its entirety.
  • Some therapies may involve a triple combination having a first antibiotic inhibiting the large mitochondrial ribosome (such as, for example, members of the erythromycin family), a second antibiotic inhibiting the small mitochondrial ribosome (such as, for example, members of the tetracycline family), administered with a pro-oxidant or an agent inducing mitochondrial oxidative stress (e.g., low concentrations of Vitamin C, radiation therapy, among other examples).
  • International Application No. PCT/US2018/028587 filed December 16, 2019, incorporated by reference in its entirety, describes further examples.
  • a fourth category of mitochondria biogenesis inhibitors are mitoketoscins, non-carcinogenic compounds that bind to at least one of ACATl/2 and OXCTl/2 and inhibit mitochondrial ATP production. These compounds are described more fully in International Application PCT/US2018/039354, fded June 25, 2018, incorporated by reference in its entirety. Mitoflavoscins and mitoflavins are a fifth category of mitochondrial biogenesis inhibitors that may be used under the present approach. These compounds are described more fully in International Patent Application PCT/US2018/057093, filed October 23, 2018 and incorporated by reference in its entirety.
  • Mitoflavoscins are compounds that bind to flavin-containing enzymes and inhibit mitochondrial ATP production.
  • Diphenyleneiodonium chloride (DPI) is an example of a mitoflavoscin.
  • a sixth category of mitochondria biogenesis inhibitors are TPP-derivative compounds that show not only a strong preference for uptake in cancer cells (bulk cancer cells, cancer stem cells, and energetic cancer stem cells), but also disrupt mitochondrial biogenesis in these cells. These TPP-derivative compounds are described more fully in International Patent Application PCT/US2018/062174, filed November 21, 2018, which is incorporated by reference in its entirety.
  • Repurposcins are a seventh category of mitochondrial inhibitors that may be used in embodiments of the present approach.
  • International Patent Application PCT/US2018/062956 filed November 29, 2018 and incorporated by reference in its entirety, describes these compounds more fully.
  • the inhibitor compound may be a myristol derivative of 9-amino-Doxycy cline.
  • the inhibitor compound may have the general formula:
  • R comprises a C4-C18 alkyl, or a pharmaceutically acceptable salt thereof.
  • Ris 13 For example, in a preferred embodiment, Ris 13.
  • An eighth category of mitochondrial inhibitors that may be used in the present approach are MDIVI-1 derivatives, as described in International Patent Application PCT/US2018/066247, filed December 18, 2018 and incorporated by reference in its entirety.
  • Mitochondrial division inhibitor- 1 (mDIVI-1) is a small molecule that selectively and reversibly inhibits DRPl. It should be appreciated that other mitochondrial inhibitors may be used, without departing from the present approach. [0069] The following paragraphs describe the materials and methods used in the experiments and results described above.
  • MDA-MB-231 cells a human breast cancer cell line, were obtained from the American Type Culture Collection (ATCC).
  • K-M analyses To perform K-M analysis on gene transcripts, the inventors used an open-access online survival analysis tool to interrogate publically available microarray data from up to 3,951 breast cancer patients (18). This allowed the inventors to determine their prognostic value. For this purpose, the inventors primarily analyzed data from ER(+)s and ER(-)/basal patients. Biased array data were excluded from the analysis. This allowed us to identify mitochondrial gene transcripts, with significant prognostic value. Hazard-ratios were calculated, at the best auto-selected cut-off, and p-values were calculated using the Log-rank test and plotted in R.
  • K-M curves were also generated online using the K-M-plotter (as high-resolution TIFF files), using univariate analysis: https://kmplot com/anal vsss/index.phj3?p :::: seivice&cancer ::: breast
  • the present approach provides gene signatures and uses thereof as a diagnostic or prognostic platform for use in conjunction with cancer treatments and therapies, to identify candidates for mitochondrial inhibitor therapy.
  • Some embodiments may take the form of a companion diagnostic, such as a diagnostic assay or test in an assayable format.
  • Example formats include a microarray or multiplex arrangement of detectable probes or ligands.
  • a companion diagnostic involving one or more of the unique gene signatures described herein, may be used as indicative of gene expression profiles of a patient’s cancer or tumor samples that may be sensitive to mitochondrial inhibitor therapy.
  • the present approach thus provides guidance about how a patient may respond to mitochondrial inhibitor therapy, and in particular how the patient’s likelihood of cancer recurrence or transmission will respond to mitochondrial inhibitor therapy.
  • assaying or testing a patient’s cancer or tumor sample for the expression of genes in one or more of the disclosed gene signatures may be followed by administering a mitochondrial inhibitor to the patient if differential expression of the genes within one or more of the disclosed gene signatures, relative to a control, is detected.
  • control may depend on the type of sample and assay, and therefore the present approach is not intended to be limited to a particular control. For example, whether the sample is isolated tumor cells or tumor tissue biopsy sample, the type of assay performed.
  • the control may vary depending on such factors.
  • a control can include an assay of normal or non-cancer cells from the patient, or from non-cancer patients.
  • normalization particularly for microarray assay platforms, may be performed to adjust for effects arising from variation in the microarray technology, rather than from biological differences between the samples, such as RNA samples, or between the addressable probes.
  • a gene signature expression profile can be prepared directly from a cancer patient’s tumor samples or specimens. This may include, for example, extracting or isolating nucleic acid, such as RNA (mRNA), or encoded protein, directly from the tumor samples or specimens (e.g., biopsied samples and specimens) and assaying for the differential expression of genes in the gene signatures, or proteins encoded therefrom.
  • mRNA RNA
  • encoded protein e.g., RNA
  • a determination of differential expression of the gene signature genes, or encoded protein, compared to a control indicates whether the patient is a candidate for mitochondrial inhibitor therapy.
  • the resulting gene signature expression profile whether prepared directly from a patient's cancer or tumor specimen or prepared from cells derived or cultured therefrom, contains transcript levels or expression levels of genes in the gene signatures of the invention, or encoded proteins thereof, that predict sensitivity of a cancer or tumor to mitochondrial inhibitor therapy, and more particularly, sensitivity of the likelihood of tumor recurrence or metastasis to mitochondrial inhibitor therapy.
  • an increased differential expression of the genes in the gene signature, relative to a control indicates that the patient is a candidate for mitochondrial inhibitor therapy.
  • Some embodiments may take the form of a method in which a gene expression dataset (e.g., a list of gene expression levels) having a gene expression level for each gene in one or more of the disclosed gene signatures, is obtained.
  • the expression levels of the genes in the dataset are compared to gene expression levels of the same genes in a control.
  • the difference in the gene expression level of the genes in the dataset compared with the control gene expression level of the same genes, if any, is calculated.
  • the patient may be identified as a candidate for mitochondrial inhibitor therapy if there is a difference in the dataset expression levels compared to the control expression levels of the same genes, or to the normalized value, for example, if the sensitivity score or cutoff value of the expression of genes in the dataset is above a threshold or cutoff value.
  • a pharmaceutically effective amount of a mitochondrial inhibitor may be administered to candidates for mitochondrial inhibitor therapy may.
  • the following paragraphs describe pharmaceutical compositions and mitochondrial inhibitor treatment.
  • This disclosure is not intended to be limited to a specific pharmaceutical formulation or pharmaceutically effective amount, at least because the effective amount depends on the mitochondrial inhibitor selected. The inventors consider these variables to be within the ordinary skill in the art, and that the routine experimentation needed to determine the pharmaceutically effective amount is not undue experimentation.
  • the demonstrative inhibitor compounds are available in various forms. For example, a Mitoriboscin compound or a Bis-TPP or d-TPP, the compound can be administered orally as a solid or as a liquid.
  • the mitochondrial inhibitor can be administered intramuscularly, intravenously, or by inhalation as a solution, suspension, or emulsion.
  • mitochondrial inhibitor (which, for the avoidance of doubt, includes salts thereof) can be administered by inhalation, intravenously, or intramuscularly as a liposomal suspension.
  • the active compound or salt can be in the form of a plurality of solid particles or droplets having any desired particle size, and for example, from about 0.001, 0.01, 0.1, or 0.5 microns, to about 5, 10, 20 or more microns, and optionally from about 1 to about 2 microns. It should be appreciated that the particular form of administration may vary, and that parameters outside of the scope of this disclosure (e.g., manufacturing, transportation, storage, shelf life, etc.) may be determinative of the common forms and concentrations of the mitochondrial inhibitor compound.
  • compositions of the present approach include a mitochondrial inhibitor (including salts thereof) as an active compound, in any pharmaceutically acceptable carrier.
  • water may be the carrier of choice for water-soluble compounds or salts.
  • organic vehicles such as glycerol, propylene glycol, polyethylene glycol, or mixtures thereof, can be suitable. Additionally, methods of increasing water solubility may be used without departing from the present approach. In the latter instance, the organic vehicle can contain a substantial amount of water.
  • the solution in either instance can then be sterilized in a suitable manner known to those in the art, and for illustration by fdtration through a 0.22-micron fdter.
  • the solution can be dispensed into appropriate receptacles, such as depyrogenated glass vials.
  • appropriate receptacles such as depyrogenated glass vials.
  • the dispensing is optionally done by an aseptic method.
  • Sterilized closures can then be placed on the vials and, if desired, the vial contents can be lyophilized.
  • a second inhibitor compound such as a glycolysis inhibitor or an OXPHOS inhibitor, may co-administer a form of the second inhibitor available in the art.
  • the present approach is not intended to be limited to a particular form of administration, unless otherwise stated.
  • pharmaceutical formulations of the present approach can contain other additives known in the art.
  • some embodiments may include pH-adjusting agents, such as acids (e.g., hydrochloric acid), and bases or buffers (e.g., sodium acetate, sodium borate, sodium citrate, sodium gluconate, sodium lactate, and sodium phosphate).
  • Some embodiments may include antimicrobial preservatives, such as methylparaben, propylparaben, and benzyl alcohol. An antimicrobial preservative is often included when the formulation is placed in a vial designed for multi-dose use.
  • the pharmaceutical formulations described herein can be lyophilized using techniques well known in the art.
  • the pharmaceutical composition can take the form of capsules, tablets, pills, powders, solutions, suspensions, and the like.
  • Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch (e.g., potato or tapioca starch) and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
  • binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
  • lubricating agents such as magnesium stearate, sodium lauryl sulfate, and talc may be included for tableting purposes.
  • Solid compositions of a similar type may be employed as fdlers in soft and hard-filled gelatin capsules.
  • compositions of the presently disclosed subject matter can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
  • the second inhibitor compound may be administered in a separate form, without limitation to the form of the d-TPP compound.
  • Additional embodiments provided herein include liposomal formulations of the active compounds disclosed herein.
  • the technology for forming liposomal suspensions is well known in the art.
  • the compound is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles.
  • the active compound due to the water solubility of the active compound, the active compound can be substantially entrained within the hydrophilic center or core of the liposomes.
  • the lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free.
  • the active compound of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer that forms the structure of the liposome.
  • the liposomes that are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.
  • the liposomal formulations comprising the active compounds disclosed herein can be lyophilized to produce a lyophilizate, which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • the pharmaceutically effective amount of an active compound described herein will be determined by the health care practitioner, and will depend on the condition, size and age of the patient, as well as the route of delivery.
  • a dosage from about 0.1 to about 200 mg/kg has therapeutic efficacy, wherein the weight ratio is the weight of the active compound, including the cases where a salt is employed, to the weight of the subject.
  • the dosage can be the amount of active compound needed to provide a serum concentration of the active compound of up to between about 1 and 5, 10, 20, 30, or 40 mM.
  • a dosage from about 0.5 mg/kg to 5 mg/kg can be employed for intramuscular injection.
  • dosages can be from about 1 pmol/kg to about 50 pmol/kg, or, optionally, between about 22 pmol/kg and about 33 pmol/kg of the compound for intravenous or oral administration.
  • An oral dosage form can include any appropriate amount of active material, including for example from 5 mg to, 50, 100, 200, or 500 mg per tablet or other solid dosage form, depending on the pharmaceutically effective amount desired.
  • the pharmaceutical composition may be in a tablet, capsule, or pill.
  • the pharmaceutical composition may have a dose of the therapeutic composition from 20 mg to 500 mg.
  • the pharmaceutical composition may comprise a tablet having 200 mg of the therapeutic compound, e.g., a compound described above, such as compound 23/G4.
  • a tablet may contain a therapeutic compound content of at least about 35%, 40%, 45%, 50% or 55%, measured by w/w percentage of the therapeutic compound (as a free base) of the core tablet.
  • the tablet may have a core formed of microcrystalline cellulose, crospovidone type A, low-substituted hydroxypropylcellulose, magnesium stearate, colloidal anhydrous silica.
  • a tablet having 200 mg of the therapeutic compound may include an inner core having microcrystalline cellulose (67.44 mg), hydroxypropyl cellulose (48.12 mg), crospovidone (29.20 mg), colloidal silicon dioxide (anhydrous) (2.12 mg), and magnesium stearate (6.36 mg), and an outer core having crospovidone (12.84 mg), colloidal silicon dioxide (anhydrous) (1.06 mg), and magnesium stearate (8.46 mg).
  • a tablet may have from about 10% to about 45% (w/w) of the therapeutic compound (e.g., compound 23/G4), and preferably about 18% to about 28% of the therapeutic compound; from about 4% to about 18% water-soluble acid; from about 20% to about 75% diluent; from about 5% to about 18% disintegrant; from about 0.2% to about 10% lubricant; and, optionally, glidant from about 0% to about 5%, and from about 0% to about 15% binder.
  • the therapeutic compound e.g., compound 23/G4
  • the tablet may have a film coating.
  • the film coating may include iron oxide black, iron oxide red, soya lecithin, polyvinyl alcohol (partially hydrolysed), talc, titanium dioxide, and xanthan gum.
  • the tablet may be coated using commercially available coating premixes, depending on the desired appearance of the final tablet.
  • Opadry® Cosmetic, Harleysville, PA
  • HPMC hydroxypropyl-methylcellulose
  • Methods of treatment described herein are preferably carried out by administering a therapeutically effective amount of a selected compound, to a subject in need of treatment.
  • the candidate subject would express elevated levels of the genes in one of the gene signatures described above.
  • the compounds can be administered by a variety of routes, including orally and parenterally, and have little or no toxicity, as discussed above.
  • kits may take the form of a kit containing reagents for the detection of genes in at least one of the gene signatures described herein, and optionally instructions for use.
  • the kit may be for predicting a patient’s candidacy for mitochondrial inhibitor therapy.
  • Kits according to the present approach may include nucleic acid probes that specifically bind to nucleotide sequences corresponding to genes in one or more of the gene signatures disclosed herein.
  • kits may include antibodies or ligands that specifically bind to polypeptides or peptides encoded by the genes in one or more of the gene signatures disclosed herein, and a means of labelling the antibodies or ligands that specifically bind to the polypeptides or peptides encoded by the genes.
  • array formats are known and used in the art and can include a wide variety of different probe structures, substrate compositions and attachment technologies.
  • Expression profiles of genes within one or more of the gene signatures disclosed herein can be generated by employing reagents tailored for inclusion in the kits according to the present approach.
  • Such reagents comprise a collection of gene specific nucleic acid primers and/or probes designed to selectively detect and/or amplify gene signature genes for use in detecting gene expression levels by using any assay format, e.g., polymerase-based assays (RT- PCR, TAQMANTM), hybridization-based assays, e.g., using DNA microarrays or other solid supports, nucleic acid sequence-based amplification assays, or flap endonuclease-based assays, or other nucleic acid quantification methods.
  • assay format e.g., polymerase-based assays (RT- PCR, TAQMANTM)
  • hybridization-based assays e.g., using DNA microarrays or other solid supports, nucleic acid sequence-based amplification assays, or flap
  • the gene specific probe and/or primer collections may include only gene signature genes, or they may include probes and/or primers for additional genes. Accordingly, the probes and/or primers used in the kits embrace oligonucleotides or antisense nucleic acids that are wholly or partially complementary to the gene biomarkers from the gene signatures disclosed herein.
  • a kit of the present approach allows for the identification of candidates for mitochondrial inhibitor therapy, by a) analyzing a sample obtained from the patient for expression levels of the genes in at least one gene signature disclosed herein; and b) comparing the expression levels to control expression levels; c) identifying the patient as a candidate for mitochondrial inhibitor therapy based on the comparison.

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Abstract

Les cellules souches cancéreuses (CSC) sont responsables de la récurrence des tumeurs, des métastases à distance et de la résistance aux médicaments, chez la grande majorité des patients atteints de cancer. Il existe un besoin urgent d'identifier de nouveaux médicaments inhibiteurs mitochondriaux capables de cibler et d'éradiquer les CSC, ainsi que des diagnostics compagnons pour identifier les candidats au traitement par inhibition mitochondriale. Dans les mammosphères 3D, 25 protéines liées aux mitochondries étaient surexprimées plus de 100 fois dans une grande collection de données de profilage transcriptionnel de patientes atteintes d'un cancer du sein ER(+). Ces 25 protéines peuvent être utilisées pour dériver des signatures géniques courtes pour prédire la probabilité de métastase distante et de récurrence tumorale. Par exemple, la signature de 4 gènes (ACLY, VDAC3, HADH2 et COX6B1) peut être utilisée pour prédire la probabilité de métastases à distance, avec un rapport de risque de 1,91 (P=2,2e-08). Une quantité pharmaceutiquement efficace d'un inhibiteur mitochondrial peut être administrée à un candidat ayant une expression élevée des gènes dans une signature génique. Cinq exemples d'inhibiteurs mitochondriaux ont montré une inhibition préférentielle et sélective de métastases de cellules tumorales, sans provoquer de toxicité significative. Sur le plan mécanique, il a été démontré précédemment que les cinq inhibiteurs mitochondriaux induisent une déplétion en ATP dans les cellules cancéreuses. Les signatures génétiques composées de 6 à 9 grandes protéines ribosomales mitochondriales présentent également une valeur pronostique pour prédire les métastases à distance, la récidive tumorale et la résistance au tamoxifène, chez les patientes atteintes d'un cancer du sein ER(+) et ER(-). Les signatures génétiques divulguées peuvent être utilisées comme diagnostics compagnons pour évaluer quels patients peuvent bénéficier le plus d'une thérapie d'inhibition mitochondriale.
EP21791579.2A 2020-04-24 2021-04-23 Procédés thérapeutiques pour la prévention des métastases et des récidives tumorales Pending EP4139484A4 (fr)

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CN116437922A (zh) * 2020-10-22 2023-07-14 卢内拉生物技术有限公司 预防转移的靶向γ亚单位的线粒体ATP抑制剂

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EP3624897A4 (fr) * 2017-05-19 2021-07-14 Lunella Biotech, Inc. Diagnostic compagnon pour inhibiteurs mitochondriaux
US11561227B2 (en) * 2017-10-11 2023-01-24 Lunella Biotech, Inc. Anti-mitochondrial inhibitors for oncogenic Ras and Myc
EP3813949B1 (fr) * 2018-06-19 2024-05-29 Lunella Biotech, Inc. Cellules souches cancéreuses « énergétiques » (e-csc) : un nouveau phénotype de cellule tumorale hyper-métabolique et proliférative, mû par l'énergie mitochondriale

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